| SERS based DNA biosensors have recently gained considerable attention for detection of target gene sequences related to diseases in medicine, the food industry, the environment and agriculture. In this work, we developed three nanoparticle based SERS assays for detection of viral DNA.;First, an indirect capture model assay using Au nanoparticles was demonstrated for SERS detection of DNA derived from the West Nile Virus (WNV) RNA genome. Upon incubation with colloidal Au, hybridization complexes containing the WNV target sequence, a complementary capture oligonucleotide conjugated to a strong tethering group and a complementary reporter oligonucleotide conjugated to methylene blue (MB), a Raman label, anchors the resultant ternary complex to Au nanoparticles and positions MB within the required sensing distance for SERS enhancement. The SERS test provides a spectral peak signature profile that is capture-specific and characteristic of the Raman spectrum for MB.;The next study decribes a model paramagnetic nanoparticle (MNP) assay that is demonstrated for SERS detection of WNV target DNA sequence. Detection is based on the capture of WNV target sequences by hybridization with complementary oligonucleotide probes covalently linked to fabricated MNPs and Raman reporter tag-conjugated gold nanoparticles (GNPs) and the subsequent removal of GNP-WNV target sequence-MNP hybridization complexes from solution by an externally applied magnetic source. Laser excitation of the pelleted material provided a signature SERS spectrum which is diagnostic for the reporter, 5,5&feet;-dithiobis(succinimidy-2-nitrobenzoate) (DSNB), and restricted to hybridization reactions containing WNV target sequences.;Finally, we developed a magnetic capture-based SERS assay for the viral DNA multiplex detection in solution using Au-coated paramagnetic nanoparticles as SERS-active capture substrates and Raman tag-conjugated oligos as reporter probes. Using the DNA derived from the Rift Valley Fever Virus (RVFV) and West Nile Virus (WNV) RNA genome, WNV as the model targets, monoplex and multiplex SERS detection were designed and demonstrated. |
